| Background and objective:With the improvement of living standards, there is an upward trend in incidence of myocardial infarction. After continuous exploration, myocardial infarction mortality rate has dropped significantly, especially after the coronary intervention was extensive used,but it caused the ischemia-reperfusion injury such as myocardial cell death, hemodynamic disorder, reperfusion arrhythmia and increased incidence of endothelial dysfunction, etc.Myocardial cells after ischemia reperfusion injury will occur a series of pathological changes,such as metabolism disorders and functional disorders. First, sodium potassium ATP enzyme in myocardial cell was injuried, sodium ion accumulation within the cell, extracellular fluid move to cells due to osmotic pressure effect, lead to cellular edema and swelling. The cell ultrastructure changes gradually on the basis of pathological changes, the integrity of cell membranes and organelles membrane is destroyed, the reversible damage transform into the irreversible damage. At the same time of ischemia-reperfusion injury, microvascular endothelial cells were damaged,the substance of expansion blood vessels was reduced and the substance shrink blood vessels was increased, the blood platelet and leucocyte adsorbs on blood vessel walls result in microvascular obstruction, microvascular level without reperfusion.According to the existing research, it is considered at present that the main mechanism of ischemia-reperfusion injury is too many free radicals generated in cells and intracellular calcium overload. In ischemic myocardial cells, the mitochondrial oxidative phosphorylation function was disordered, make the mitochondrial oxidative phosphorylation pathway reduced and oxygen free radicals generation processed, and because of ischemic makes oxygen free radical scavenger activity is reduced, eventually produce a large number of active oxygen free radicals. Besides, neutrophils "breathing explosion" and the xanthine oxidase can produce oxygen free radicals. Oxygen free radicals have lively reactivity,can react with membrane phospholipid, protein, nucleic acid and so on, leading to cell damage.When cell hypoxia, cell membrane permeability increased, all sorts of ion transport pump dysfunction, leading to intracellular calcium accumulation, intracellular calcium concentration increased significantly can activate calcium dependent protease, proteolytic enzyme and phospholipase, and deposit on the mitochondria, leading to cell damage. The ischemia postconditioning is that brief,repeat opening and closing coronary when myocardial ischemia reperfusion immediately, then restore coronary artery blood flow. Studies found that ischemia postconditioning can protect endothelial cells stable, reduce the generation of oxygen free radicals in reperfusion cardiomyocytes, reduce neutrophil activation and aggregation, reduce capillary pathological changes, affect the ischemia-reperfusion injury of each link, reduce myocardial cell damage. Studies showed that ischemia postconditioning can protect cardiomyocytes in ischemia-reperfusion injury,it played a protective role in improving survival rate, reduce cardiomyocytes apoptosis, reducing myocardial infarction area and improve endothelial cell function.The ischemia postconditioning operation method is simple, mainly used in PCI in acute myocardial infarction treatment.After the clinical application,it showed good clinical effect. Garcia found that the ischemia postconditioning group had lower peak of CK and CK-MB proportion comparied with control group, and through to the ischemia postconditioning treatment, the myocardial infarction area decreased. The ischemia postconditioning both in scientific research and clinical showed good myocardial protection,it confirmed the feasibility, safety, and potential effects in clinical.Cardiotrophin-1(CT-1) was established in1995by Pennica, separated from mouse embryonic stem cells culture, to be classified in interleukin-6(IL-6) and leukemia inhibitory factor (LIF) family of cytokines,its chemical structure was similar with interleukin-6and could be combined with gp130receptor. In the early days, CT-1was a marker of myocardial disease diagnosis and prognosis. Recent studies have found that it is a kind of important neural hormone in the myocardial injury, it is mainly expressed in myocardial cells and fibroblasts, it has the functions to promote cell survival and normal development of the heart, it also expressed in skeletal muscle, liver, ganglion and so on. Animal experiments in rats, the CT-1mRNA expression increases in the early stages of ventricular hypertrophy, and the CT-1has maintained a high level expression after myocardial hypertrophy.Experiments have demonstrated that CT-1expression increased significantly in ischemia myocardium after myocardial infarction, reduce the cardiomyocyte death, promote residual cardiomyocytes survive, accelerate the healing of infarction area. Basic research found that CT-1can promote the newborn rat cardiomyocyte survive in serum free medium, the above indicate that CT-1plays a protective role on ischemia anoxic cardiomyocytes. Lopez B found that plasma CT-1level was positively correlated with the degree of heart failure, left ventricular mass index, negative correlation with the ejection fraction,and independence between many complex potential influencing factors, prompt that CT-1can be used as indicators to evaluate the degree of heart failure. Further research shows that CT-1was more sensitive to the diagnosis of C stage heart failure patients compaired with N end brain natriuretic peptide, the measurement of plasma CT-1concentrations provide additional information to C stage heart failure patients.CT-1is not only closely related to ventricular remodeling, but also affect the blood flow dynamics. Jin found that rats given intravenous CT-1would happen systemic hypotension, and the blood pressure effect was dose dependent. Further study found that the mechanism of CT-1decrease blood pressure was to decrease peripheral vascular resistance,at the same time CT-1increased heart rate and cardiac output, nitric oxide synthetase inhibitors can be reduced with faster heart rate, blood pressure effect, suggest that NO could adjust CT-1influence on hemodynamics. Experiments have demonstrated that CT-1expression increased significantly in ischemia myocardium after myocardial infarction (MI), reduce the myocardial cell death, promote residual myocardial cells survive, accelerate the healing of infarction area. Basic research found that CT-1can promote the newborn rat myocardial cells in serum free medium, above indicate that CT-1plays a protective role on myocardial ischemia anoxic cells.After the cardiomyocyte suffer from ischemia-reperfusion injury, mitochondriai permeabiiity transition pore(mPTP) permeability increase, the promote apoptosis protein released from mitochondria to the cellular plasma, leading to cell death--apoptosis or necrosis.The postconditioning can active ERK1/2pathway, make its downstream of GSK-3beta phosphorylation and lose activity.It prompted that ERK1/2signal pathway activation inhibits mPTP open, reduce the cell apoptosis and necrosis.The activation of ERK1/2signaling pathways can also suppress BAD activity or facilitate the transcription of the protein and play a protective role. CT-1 has the myocardial protection effects,but the specific mechanism of action and signal transduction pathways still needs further research to confirm.The H9C2cardiac muscle cell lines is as as experiment object, under the condition of in vitro,used hypoxia reoxygenation model simulated ischemia-reperfusion injury,used hypoxia postconditioning model simulated ischemia postconditioning, combined CT-1and hypoxia postconditioning, through the observation of the myocardial cell survival rate, apoptosis rate, Bad mRNA expression and phosphorylation ERK1/2protein(p-ERK1/2protein) expression changes,to identify the myocardial protective effect in hypoxia reoxygenation on the basis of combined CT-1and hypoxia postconditioning.The application of signaling pathway blockers could block the signal transduction, further clarify the myocardial protection mechanism and signal transduction pathways of CT-1and hypoxia postconditioning to hypoxia reoxygenation.Materials:The H9C2cardiac muscle cell lines culture in vitro. The H9C2cardiac muscle cell lines culture in DMEM-high sugar medium containing10%fetal bovine serum and37℃and5%CO2incubator,use the index phase of cells of recovery after2,3generation.Method:Cells can be divided into6groups and given different treatment respectively, a. Normal control group:cells culture in DMEM-high sugar medium containing10%fetal bovine serum without any processing.b. Hypoxia reoxygenation group:hypoxia, cells cultured in pH6.8D-Hanks culture fluid in the hypoxia incubator (95%N2and5%CO2,37℃) training3h; reoxygenation,turn D-Hanks culture fluid to DMEM containing10%fetal bovine serum after hypoxia3h, reoxygenation3h in MCO-17AIC02incubator (95%02,5%CO2,37℃).c. Hypoxia reoxygenation+hypoxia postconditioning group:the same as group2,5min reoxygenation/5min hypoxia before reoxygenation for3cycles.d. Hypoxia reoxygenation+hypoxia postconditioning+CT-1group:the same as hypoxia reoxygenation group, add10ug/1CT-1after hypoxia for3h, apply5min reoxygenation/5min hypoxia for3cycles,and then reoxygenation3h.e. Hypoxia reoxygenation+hypoxia postconditioning+CT-1+ERK inhibitors group:add20umol/1ERK inhibitors30min before hypoxia finish, add10ug/1CT-1after the completion of hypoxia,then apply5min reoxygenation/5min hypoxia for3cycles, reoxygenation3h.f. Hypoxia reoxygenation+hypoxia postconditioning+dimethyl sulfoxide group:ERK inhibitors turn to dimethyl sulfoxide, other are the same as group5. Apply the MTS method to detect each group survival rate, to detect cell apoptosis rate by flow cytometry instrument, using Western Blot to detect the expression of cells regulate protein kinase (ERK1/2and p-ERK1/2), using Q-PCR to detect the expression of Bad mRNA.Result:Compared with control group, the hypoxia reoxygenation group, hypoxia reoxygenation+hypoxia postconditioning group, hypoxia reoxygenation+hypoxia postconditioning+CT-1group, hypoxia reoxygenation+hypoxia postconditioning+CT-1+ERK inhibitors group and hypoxia reoxygenation+hypoxia postconditioning+dimethyl sulfoxide group have lower cell survival rate,higher apoptosis rate, Bad mRNA expression increased, P<0.05, differences were statistically significant. After hypoxia postconditioning processing, the cells survival rate increase, apoptosis rate decrease, p-ERK1/2protein expression increase, Bad mRNA expression decrease compared to hypoxia reoxygenation group, P<0.05, the difference is statistically significant.Add CT-1on the basic of hypoxia postconditioning processing, the cells survival rate increased, apoptosis rate decreased, p-ERK1/2protein expression increased, Bad mRNA expression decreased, P<0.05, the difference is statistically significant. After add ERK inhibitors in hypoxia reoxygenation+hypoxia postconditioning+CT-1group, the cells survival rate decreased, apoptosis rate increased, p-ERK1/2protein expression decreased, Bad mRNA expression increased, P<0.05, the difference is statistically significant. Due to ERK inhibitors dissolved by dimethyl sulfoxide, and dimethyl sulfoxide has certain cytotoxicity, ERK inhibitors turn to dimethyl sulfoxide, compared to hypoxia reoxygenation+hypoxia postconditioning+CT-1group, tne cell survival,apoptosis rate, p-ERK1/2protein expression and Bad mRNA expression has no obvious change, P>0.05. ERK1/2expression of each group did not see obvious difference, p>0.05.Conclusion:Hypoxia reoxygenation can obviously injury myocardial cells, hypoxia postconditioning processing can reduce the myocardial cell injury by hypoxia reoxygenation, combined CT-1can significantly enhanced protection, its mechanism may be that active ERK1/2signaling pathways, make ERK1/2protein phosphorylation, which cuts Bad mRNA expression, reduces myocardial cell damage. |
PDF Full Text Request